Determination Of 1-Deoxynojirimycin In Silkworm And Mulberry And Optimization Of Extraction Process

In order to study the variation of 1-Deoxynojirimycin (DNJ)content in different mulberry varieties leaves and silkworm varieties, to screen silkworm varieties with better enrichment ability from the forage mulberry leaves, 10 mulberry varieties and 18 silkworm varieties were used as the materials for determination DNJ contents in whole body powder of day 3 silkworm larvae of the 5th instar by reverse-phase high-performance liquid chromatography-ultraviolet spectrometry(RP-HPLC-UV), optimization of extraction process using central composite design-response surface methodology(CCD-RSM), so as to exploring sericultural resources are used to functionnal fooding and medicine study for providing theoretical basis. The main results are as follows:1. The method of RP-HPLC-UV. In potassium borate buffer (pH=8.5), FMOC-Cl was added to derive DNJ. The DNJ-FMOC was produced which can absorb ultraviolet radiation and then was tested with ultraviolet radiation inspector. The results showed that the peak area of DNJ-FMOC was correspond with the concentration of DNJ. All of the precision test, recurring test, stability test and the recovery test showed that the method was credible and stable, and it could be used as an effective method in determining the DNJ content in mulberry leaves, silkworm and other related health products.2.The DNJ content in 10 mulberry varieties leaves in Spring and Autumn were determined by RP-HPLC-UV. The rusults showed that The DNJ content in Shansang305 mulberry leaves in Autumn was the highest, DNJ mass fraction was 0.3795%, had extremely significant difference with other mulberry varieties leaves(P<0.01), it indicated that the DNJ content had remarkable difference in different mulberry varieties. Determinating DNJ content in the same mulberry variety in different collection seasons, the rusults showed that the mulberry leaves of 707, Tengsang, Puxuanyihao, Shansang305, Shansang306, Shansang402 in Autumn had extremely significant difference with DNJ content in spring(P<0.01), it indicated that the DNJ content had remarkable difference in the same mulberry variety in different collection seasons. Determinating DNJ content in different genome number of mulberry varieties(diploid, triploid and tetraploid) the rusults showed that the DNJ content in triploid mulberry in Autumn was the highest, DNJ mass fraction was 0.3439%, had extremely significant difference with diploid and tetraploid(P<0.01), it indicated that the DNJ content had remarkable difference in different genome number of mulberry varieties.3. The DNJ contents in whole body powder of day 3 silkworm larvae of the 5th instar in 18 silkworm varieties were determined by RP-HPLC-UV. The rusults showed that silkworm variety 797 of Chinese strain had the best enrichment result to DNJ from mulberry leaves, DNJ mass fraction in the whole body powder reached 0.4345%, had extremely significant difference with other silkworm varieties (P<0.01), it indicated that different silkworm varieties had remarkable different enrichment ability to DNJ from forage mulberry leaves. Determinating DNJ content in silkworm varieties different geographical strains(Japanese strains, Chinese strains and European strains), The rusults showed that Silkworm varieties of Japanese and European strains had significantly different enrichment ability to DNJ from forage mulberry leaves (DNJ mass fraction of the former was 0.3007% and that of the latter was 0.2469%). The average DNJ mass fraction in silkworm varieties of Chinese strain was 0.2880%, having no significant difference with those of Japanese and European strains. Silkworm varieties of different moltinisms (trimolter or tetramoulter) had no remarkable difference in their enrichment ability to DNJ from forage mulberry leaves. The correlation between DNJ mass fraction in the whole body powder and DNJ content in mulberry leaves were analyzed by feeding larvae of 6 silkworm varieties with mulberry leaves of different varieties. The result showed that the correlation coefficient was 0.8260. The combination with the highest DNJ content in the whole body powder was silkworm variety Wu2 and mulberry variety Shansang403, followed by silkworm variety Wugongtuzhong and mulberry variety Tiansang, silkworm variety 798 and mulberry variety Shansang403.4. In order to optimize the DNJ extraction process from mulberry leaves, central composite design-response surface methodology was used to optimise DNJ extraction yield. Four independent variables, extraction concentration, temperature, time and liquid-solid ratio were investigated to obtain the optimum extraction yield. Established preliminarily the model of quadratic polynomial and definited the optimum conditions. The result showed that optimum conditions of DNJ extraction from mulberry leaves were: 70% ethanol, 70°C, 4h for extraction time and 32.5 at liquid-solid ratio. Under the optimized condition the predict DNJ extraction yield was 0.247%, the actual DNJ extraction yield was 0.263%, bias was -6.478%. It indicated that the model was predictive.5. In order to optimize the DNJ extraction process from silkworm powder, central composite design-response surface methodology was used to optimise DNJ extraction yield. Four independent variables, extraction concentration, temperature, time and liquid-solid ratio were investigated to obtain the optimum extraction yield. Established preliminarily the model of quadratic polynomial, definited the optimum conditions. The result showed that optimum conditions of DNJ extraction from silkworm powder were: 80% ethanol, 75°C, 4h for extraction time and 32.5 at liquid-solid ratio. Under the optimized condition the predict DNJ extraction yield was 0.339%, the actual DNJ extraction yield was 0.327%, bias was 3.540%. It indicated that the model was predictive.